Optical assay for stored human platelets

ABSTRACT

The viability of a pack of stored blood platelets is monitored by gripping the pack between two plates (6) and (7) which are closed to pinch together the walls of the bag (10) along an L-shaped seal (14), leaving a channel (17), squeezing a part (15) of the bag by means of a reciprocating plunger (19), so that the platelets continually flow to and fro between the part (15) and the part (16), through the channel (17), and passing a beam of light from an LED (27) through the channel (17), to a photoresistor (29). The AC signal from the photoresistor corresponds to the fluctuations in the intensity of the light passing through the channel (17) and the amplitude of this signal is representative of the viability, and hence the clinical acceptability, of the platelets.

BACKGROUND OF THE INVENTION

Concentrates of blood platelets in plasma are infused into patients totreat bleeding problems. Human platelets can withstand storage for onlyup to five days before progressive loss of platelet viability negatesfurther clinical use. The lifetimes of stored platelets are highlyvariable, so it is inevitable that some useless platelets are given topatients while other packs of viable platelets are disposed ofneedlessly at the end of their arbitrary shelf life. Unfortunately,there is not in use any test of platelet viability which can be usedwithout removing a sample of platelets from the pack. This isinconvenient, time consuming and involves the risk of the pack contentsbecoming infected.

Recently, an optical technique has been proposed for the measurement ofplatelet viability without withdrawing samples from the pack. Thetechnique depends on the light scattering properties of the plateletswhich differ when the platelets are functional or dead. It is believedthat this effect stems from the change of shape of platelets duringstorage. Functional platelets have a discoid shape. As the plateletsage, more and more of them lose their discoid shape and become nearlyspherical, thereby changing their light scattering property. Thepreviously proposed technique has involved a comparison of the lightscattering properties of the platelets when they are flowing and whenthey are stationary. Thus, when a beam of light is incident on a pack ofthe platelets, it has been found that the ratio of the intensity oflight scattered in a particular direction when the platelets are moving,divided by the intensity of light scattered in the same direction whenthe platelets are still, may be less than 0.7 when the platelets arefunctional but approaches unity as the platelets die. This technique isof theoretical interest but its application would be difficult inclinical use as it would be necessary to remove a pack of platelets fromthe rocking tray, on which such packs are normally stored to keep theplatelets in motion, and to transfer it to some device in which thelight scattering properties could be measured both when the plateletsare in motion and stationary.

SUMMARY OF THE INVENTION

A further characteristic of the optical properties of the platelets hasnow been appreciated by one of the present inventors. This is that theamplitude of fluctuations of the fluctuating light transmitted throughthe sack of moving platelets in a particular direction remainssubstantially constant for up to a day when the platelets are fresh, butthen gradually decays nearly to zero as the platelets die. The meanamplitude of the transmitted light varies from sample to sample but inall cases the decay in the amplitude of the fluctuations follows asimilar curve.

This novel appreciation leads to a method of monitoring non-invasivelythe viability of a pack of stored platelets, the method comprisingagitating the pack, irradiating the pack with a beam of light, anddetecting the amplitude of the fluctuations in the intensity of lighttransmitted through the pack of platelets in a particular direction andcomparing this with a datum related to the amplitude of the fluctuationsin the intensity of light transmitted in the same direction from asimilar pack of fresh platelets under similar conditions.

A blood platelet monitoring assembly for carrying out the new methodcomprises at least one translucent pack of blood platelets; means foragitating the pack; a light source for irradiating the pack, while thepack is being agitated, with a beam of light; a photoelectric elementfor collecting light transmitted through the pack in a particulardirection; and means responsive to an AC component, corresponding tofluctuations in the intensity of the transmitted light, in the outputsignal from the photoelectric element.

The particular advantage of the new method is that the viability of thenew platelets can be monitored while the packs are being agitated, whichis the accepted condition in order to prolong the life of the platelets.

Preferably, the pack is agitated to cause the platelets to flowcontinually to and fro across a zone through which the pack isirradiated with the light beam. The agitation could be provided byrocking the pack, for example in association with a tray similar tothose on which platelet packs are conventionally stored. The frequencyof the fluctuations in the amplitude of the transmitted light will betwice the frequency of the oscillating flow.

In the usual case in which the pack comprises a bag with two major,generally translucent, opposed walls, the bag may be supported so thatthese walls are pinched together to divide the interior of the bag intotwo parts connected by a channel constituting the zone; one of the partsbeing periodically squeezed to cause the to and fro flow through thechannel. The channel is then preferably positioned adjacent to thebottom of the bag, as oriented in its support, so that any tendency ofthe platelets to sediment at the bottom of the bag is avoided by thecontinual disturbance of the platelets by the oscillating flow.

The earlier proposed optical technique used, as a light source, a bulkylaser which was housed, together with plates between which the packunder test was sandwiched, in a light proof housing. The laser beam wasdirected through windows in the plates, and through the interveningpack, to the photoelectric element. This is all unacceptably cumbersomefor clinical use. In order to carry out the new method, it is nowproposed to utilise a device comprising two surfaces between which thepack of platelets under test is gripped, one of the surfacesincorporating a light source, such as light emitting diode, and theother incorporating, in a position opposite the light source, aphotoelectric element, such as a photodiode or a photoresistor. Thisdevice is extremely compact, requires no screening from exterior light,and ensures that the optical system is correctly aligned as soon as thepack is gripped between its surfaces.

The datum reading might be read by a technician and recorded when thepack is fresh, preferably within twelve hours of the platelets havingbeen obtained from a human body. Prior to clinical use, a second readingwould be taken by the technician, and the platelets may then beconsidered to be sufficiently non-functional for clinical use, if theamplitude of the fluctuations has decreased to say 25% or less of thedatum figure for fresh platelets. An appropriate display would thenmerely need to provide a numerical reading representing the amplitude ofthe AC signal. Alternatively, appropriate circuitry may incorporate amicroprocessor or other intelligent circuit which stores the datumsignal corresponding to the pack when fresh, and provides a continuousdisplay representing the ratio of the current signal to the datumsignal. In the simplest case, this may be a green light which isilluminated when the platelets are still functional and a red light whenthey are not.

Surprisingly, a datum reading for each pack when fresh may beunnecessary. This is because, first, until the number of live plateletshas dropped to a very low level, the amplitude of the fluctuations inthe transmitted light is almost independent of the density or volume oflive platelets in the pack, and, secondly, because the reduction in theamplitude of the fluctuations diminishes very quickly when anappreciable proportion of the platelets become non-viable. It followsthat, for a particular optical system, infrequent calibration is allthat is necessary to provide a preset threshold corresponding to theamplitude of the fluctuations in the intensity of the transmitted light,below which there are insufficient living platelets for the pack to beclinically functional. The light or other display can then be arrangedmerely to indicate whether the amplitude of the AC signal is above orbelow the preset threshold.

BRIEF DESCRIPTION OF THE DRAWINGS

An example of an optical assay system in accordance with the inventionis illustrated diagrammatically in the accompanying drawings, in which:

FIG. 1 is an exploded, partially cut away, perspective view;

FIG. 2 is a perspective view of a plate of a pack supporting device;

FIG. 3 is an elevation of the system; and,

FIGS. 4 and 5 are relevant graphs.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

As shown in FIG. 1, the system incorporates a device comprising a first,vertical fixed plate 6, and a similar plate 7 which is connected to itby a hinge 8. Each plate consists of a peripheral frame formedintegrally with a central cruciform portion defining with the peripheralframe, four windows. In each case the upper windows are open. The lowerwindows of the plate 7 and one of the windows of the plate 6 arepartially closed by screens 9. The plate 7 is arranged to be swung upgenerally parallel to the plate 6, about the hinge 8, to grip betweenthe two plates a conventional bag 10 of blood platelets in plasma, Thebag has, in a peripheral seam, holes 11 which are fitted onto pegs 12projecting from the plate 7. The two plates 6 and 7 are held closed,with the bag 10 gripped between them, by means of side catches 13. FIG.1 shows the bag in full lines separate from the plates and, in chaindotted lines, in position on the plate 7.

With the bag positioned between the plates an L-shaped seal 14 pinchesthe bag along a pinch line shown at 14' in dotted lines in FIG. 1, todivide the interior of the bag into parts 15 and 16, which areinterconnected by a channel 17 adjacent to the bottom of the bag.

Mounted on a common base with the plate 6 is a cylindrical housing 18 inwhich there slides axially a plunger 19 carrying at one end a disc 20and at the other end a disc 21. A helically coiled compression spring 22acts between the disc 21 and an end wall of the housing 18 to urge thedisc 20 to a withdrawn position away from the plate 6. A cam 23, whichis fixed on a shaft 24, driven at constant rotational speed by anelectric motor 25, periodically moves the plunger 19 and hence the disc20 through an open lower window 26 in the plate 6, against the action ofthe spring 22, to squeeze the part 15 of the bag 10. This displaces thecontents of the bag, through the channel 17 up into the part 16. As thecam 23 rotates to its smaller radius orientation relative to the disc21, the plunger 19 and disc 20 are able to move back out of the plate 6under the action of the spring 22, thus releasing the squeeze pressureon the part 15 of the bag 10. The differential hydrostatic pressure atthe channel 17, resulting from the previous displacement of the contentsinto the part 16 of the bag 10, then causes the contents to flow backthrough the channel 17 into the part 15. This periodic rotation of thecam 23 thus causes to and fro flow in the bag 10 through the channel 17,as indicated by the arrows, and keeps all the contents in continualmotion. The frequency of the cam 23 may be about 2 Hz and the stroke ofthe disc 20 about 3 ml.

In alignment with the channel 17, the plate 6 carries a cylindricalmounting 27, incorporating a light emitting diode (Hewlett-Packard HLMP3750, wavelength 635 nm), which is energised through a line 28. The LEDdirects its beam through an opening 36 in the plate 6, and through thechannel 17 in the bag 10, where it is scattered by the platelets. Anylight which is ultimately transmitted in the straight ahead direction,passes through a complimentary opening in the plate 7, and is picked upby a light sensitive resistor in a cylindrical mounting 29. Theelectrical output of the photoresistor, which will correspond directlyto the amplitude of the light transmitted at an angle of substantially0° to the beam from the LED, is transmitted along a line 30 to amicroprocessor 31. This microprocessor deduces the AC component in thesignal by sensing and subtracting the maximum and minimum signal levels.

As platelets in the bag 10 are caused to flow with an oscillating motionto and fro through the channel 17, the platelets will, at maximumvelocity, be caused to stream in mutual alignment through the channel,and, upon reversal of the direction of flow, will adopt a relativelyrandom orientation. This is what leads to the fluctuations of theamplitude in the intensity of light received from the LED by thephotoresistor.

A display of the microprocessor 31 may be preset to respond to an ACsignal having an amplitude above or below a certain thresholdcorresponding to an acceptable or unacceptable number of viableplatelets in the bag 10, respectively. Alternatively, if a pack istested when the platelets are fresh, the microprocessor 31 may beinstantly calibrated to a corresponding threshold, by recording thedatum reading by, for example, depression of a switch 32. In eithercase, when the amplitude is above the threshold, indicating that theplatelets are viable, a green lamp 33 may be illuminated. When the bagof platelets has become effectively non-functional, the amplitude willfall below the threshold, and a red lamp 34 will be illuminated. In thisway any particular bag of platelets can be used up to a last possiblemoment when a satisfactory proportion of its platelets are viable forclinical use.

FIG. 4 shows a graph of the variation in a voltage V_(osc) correspondingto the amplitude of the AC component in the signal from thephotoresistor, with a period of days. The sharp cut off after, onaverage, five days, is very noticeable.

FIG. 5 shows a variation in a similar voltage with a variation in thedensity of viable platelets in the bag 10. This shows that the amplitudeof the AC signal is substantially independent of the density when thedensity is between 300 and 700 times 10⁹ /1, and falls off very sharplyat lower densities. Although variations in the density and indeed in thevolume of platelets in a typical bag 10 occur, dependent upon thefraction of the centrifuged blood which is encapsulated in that bag, anybag of viable platelets can be expected to have a density within thisrange 300 to 700.

The walls of the bag 10 are conventionally of calendered plastics whichis translucent but the wall surfaces are rough and may produce unwantedscattering of the light beam, even to the point of obscuring thescattering by the platelets. The plasma wets the inner surfaces of thebag walls but the problem remains at the outer wall surfaces. Toovercome this and provide an optically clear window through each bagwall, a patch of optically clear tape 35 with a coating of an opticallyclear adhesive is stuck on the outer surface of each bag wall inalignment with the channel 17.

A bank of illustrated devices may be set up alongside one another anddriven via a common camshaft 24, and may be coupled to a common signalprocessor and display.

What is claimed is:
 1. A device for use in a blood platelet monitoringassembly of the kind comprising at least one translucent pack of bloodplatelets comprising:means for periodically agitating the pack to causeplatelets in the pack to flow, said means for agitating including firstand second surfaces between which the pack of platelets is adapted to begripped, light source means in one of said surfaces for irradiating thepack with light and causing light to be transmitted through the pack ina given direction, photoelectric means in the other of said surfaces ina position opposite to said light source means for collecting the lighttransmitted in said given direction and producing an electrical signalhaving an AC component with an amplitude equal to the difference betweenminimum and maximum levels of transmitted light, said AC componenthaving a frequency equal to the frequency of periodic agitation, andmeans responsive to said signal for extracting the AC component thereof,comparing the amplitude of the AC component with a reference datum andgenerating a visual display indicating one of acceptability ornon-acceptability of the viability of the pack.
 2. A device according toclaim 1, wherein said surfaces are the inner faces of two hinged plates.3. A device according to claim 1, wherein said surfaces are shaped topinch together opposed walls of said pack to divide the interior of saidpack into two parts connected by a channel situated between said lightsource and said photoelectric element; and means are provided forperiodically squeezing only one of said parts and alternately releasingsaid one of said parts without pressing the other of said parts to causesaid platelets in said pack to flow to and fro through said channel. 4.A device according to claim 1, wherein said light source is a lightemitting diode and said photoelectric element is a photoresistor.